Antibiotic resistance has been declared one of the three greatest threats to human health by the World Health Organization. Infections caused by multi-drug resistant (MDR) bacterial pathogens create substantial health and economic impacts on society due to the lack of effective therapeutic options created by the rapid acquisition of resistance and stagnant antibiotic development programs. This lack of treatment options is particularly relevant for MDR Gram-negative pathogens, such as Pseudomonas aeruginosa, Acinetobacter baumanii and Klebsiella pneumoniae, which have shown a great propensity to thwart antibiotic treatments and standard hospital disinfection procedures. Agile Sciences is developing a novel class of small molecules, based on a 2-aminoimidazole (2-AI) scaffold, that substantially increase the susceptibility of MDR bacteria to antibiotic therapies. As an adjuvant therapy to current antibiotics, the 2-AI molecules have the potential to provide a much improved treatment option for MDR Gram-negative bacterial infections. Phase I equivalent work has displayed the potential of 2-AI compounds as antibiotic potentiating agents against MDR Gram-negative bacterial pathogens. Our efforts have shown that: 1) two lead compounds, AGL- 503 and AGL-553, are able to lower the antibiotic MIC values against MDR Gram-negative bacteria; 2) an AGL-503-meropenem combination therapy has the ability to enhance survival and decrease bacterial burden (compared to meropenem alone) in a MDR P. aeruginosa acute lung infection model; 3) 2-AI compounds possess favorable safety and pharmaceutical profiles; and 4) 2-AI compounds act via a novel mechanism of action that retards antimicrobial resistance. Collectively, this data provides strong support for a continued development program to define the potential therapeutic utility of the 2-AI class of molecules as an antibiotic combination therapy for treating infections caused by MDR Gram-negative bacteria. In Phase II, we will focus development of the 2-AI class of molecules to address the substantial unmet need posed by MDR Gram-negative bacterial infections. First, in Aim 1, we will identify the optimal antibiotic combinations for the lead 2-AI compounds against P. aeruginosa, K. pneumoniae, and A. baumanii. The optimal combinations will be evaluated for pharmacodynamics using the mouse thigh infections mode. In Aim 2, the off-target effects of AGL-503 and AGL-553 will be evaluated through off-target panel screens and characterizations of target binding. The 2-AI-antibitoic combination with the most promising results in Aim 1 will be evaluated for safety and efficacy in pneumonia and urinary tract infection murine models. The efforts in Aim 3 will inform the target product profile (TPP) of the candidate compound, and preliminary scale-up work will be performed to facilitate transfer of the synthetic route of the compound to a contract manufacturing organization. At the conclusion of this two-year grant, Agile Sciences will have declared an IND candidate compound and defined the TPP so as to facilitate follow-on IND-enabling studies.